A Photobacterium sp. α2–6-sialyltransferase (Psp2,6ST) mutant with an increased expression level and improved activities in sialylating Tn antigens

•Several mutants of Photobacterium sp. α2–6-sialyltransferase (Psp2,6ST) were obtained.•A366G mutant has an increased expression level.•A366G mutant also has an improved catalytic activity in sialylating Tn antigens.•A one-pot two-enzyme sialylation system improved STn antigen synthetic efficiency.•Psp2,6ST A366G mutant is a powerful catalyst for synthesizing α2–6-sialosides.

In order to improve the catalytic efficiency of recombinant Photobacterium sp. JT-ISH-224 α2–6-sialyltransferase Psp2,6ST(15–501)-His6 in sialylating α-GalNAc-containing acceptors for the synthesis of tumor-associated carbohydrate antigens sialyl Tn (STn), protein crystal structure-based mutagenesis studies were carried out. Among several mutants obtained by altering the residues close to the acceptor substrate binding pocket, mutant A366G was shown to improve the sialyltransferase activity of Psp2,6ST(15–501)-His6 toward α-GalNAc-containing acceptors by 21–115% without significantly affecting its sialylation activity to β-galactosides. Furthermore, the expression level was improved from 18–40 mg L−1 for the wild-type enzyme to 72–110 mg L−1 for the A366G mutant. In situ generation of CMP-sialic acid in a one-pot two-enzyme system was shown effective in overcoming the high donor hydrolysis of the enzyme. Mutant A366G performed better than the wild-type Psp2,6ST(15–501)-His6 for synthesizing Neu5Acα2–6GalNAcαOSer/Thr STn antigens.

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